DNA methylation is not responsible for p21WAF1/CIP1 down-regulation in osteoarthritic chondrocytes.
OBJECTIVE
In this study, we were interested in the overall methylation level in aged and degenerated cartilage. Also, we looked at one gene which might be involved in the re-initiation of replicative activity in osteoarthritis (OA) chondrocytes, p21(WAF1/CIP1). p21(WAF1/CIP1) was previously suggested to be down-regulated in OA chondrocytes and is known to be regulated by epigenetic modulation.


METHODS
Total methylation levels were analyzed by high pressure liquid chromatography (HPLC), mRNA expression of p21(WAF1/CIP1) and DNMT enzymes by real-time polymerase chain reaction. The methylation status of the p21(WAF1/CIP1)- promotor using bisulfite genomic sequencing was evaluated.


RESULTS
General methylation analysis of genomic DNA showed no difference in between normal and aged/OA chondrocytes. Also no difference in methylation of the promotor of the p21(WAF1/CIP1) gene was detectable, which was significantly down-regulated in OA chondrocytes. DNMT1 and DNMT3a were expressed with no significant changes of expression levels found in OA chondrocytes.


CONCLUSIONS
Cell cycle progression inhibitor p21(WAF1/CIP1) is expressed in normal and significantly down-regulated in OA articular chondrocytes, which may mediate the re-initiation of cell proliferation in OA cartilage. However, the suppression of p21(WAF1/CIP1) mRNA expression is not due to hypermethylation of its promotor. No overall changes in genome methylation levels were found in aged or OA cartilage. Interestingly, significant expression of DNA methyltransferases was found in articular chondrocytes, which supports that DNA methylation could still be a relevant mechanism of gene regulation in (osteoarthritic) chondrocytes, though not on an overall genomic level nor specifically for the regulation of the p21(WAF1/CIP1) gene.
Sesselmann
S
S
Institute of Pathology, University Hospital Erlangen, Krankenhausstr. 12, 91054 Erlangen, Germany.
Söder
S
S
Voigt
R
R
Haag
J
J
Grogan
S P
SP
Aigner
T
T
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D019902:Chondrocytes / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis* / Q000235:genetics; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D015536:Down-Regulation / Q000235:genetics; D016678:Genome; D006801:Humans; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C000717967:DNMT3A protein, human; D012333:RNA, Messenger; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C000619380:DNMT1 protein, human